1887

Abstract

The Psc gene from has been cloned and expressed in the heterologous host , under the control of the methanol inducible promoter. The native laccase signal sequence was effective in directing the secretion of expressed in . The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 438. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 33, 6, 65 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 °C and pH 35. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.

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2002-12-01
2019-10-20
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References

  1. Agematu, H., Kominatu, K., Shibamoto, N., Yoshioka, T., Nishida, H., Okamoto, R., Shin, T. & Murao, S. ( 1993; ). Transformation of cephalosporanic acid into new cephalosporin derivatives. Biosci Biotechnol. Biochem 57, 1387-1388.[CrossRef]
    [Google Scholar]
  2. Alexandre, G. & Zhulin, I. B. ( 2000; ). Laccases are widespread in bacteria. Trends Biotechnol 18, 41-42.[CrossRef]
    [Google Scholar]
  3. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403-410.[CrossRef]
    [Google Scholar]
  4. Aramayo, R. & Timberlake, W. E. ( 1990; ). Sequence and molecular structure of the Aspergillus nidulans yA (laccase I) gene. Nucleic Acids Res 18, 3415.[CrossRef]
    [Google Scholar]
  5. Ausubel, F. M., Brent, R., Kingston, E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1996). Current Protocols in Molecular Biology. New York: Wiley.
  6. Baker, W. L., Subapathy, K., Vibat, M. & Lonergan, G. ( 1996; ). Laccase catalyses formation of an inadamine dye between 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylamonobenzoic acid. Enzyme Microb Technol 18, 90-94.[CrossRef]
    [Google Scholar]
  7. Berka, R. M., Schneider, P., Golightly, E. J., Brown, S. H., Madden, M., Brown, K. M., Halkier, T., Mondorf, K. & Xu, F. ( 1997; ). Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol 63, 3151-3157.
    [Google Scholar]
  8. Bourbonnais, R., Paice, M. G., Reid, I. D., Lanthier, P. & Yaguchi, M. ( 1995; ). Lignin oxidation by laccase enzymes from Trametes versicolor and the role of the mediator in 2,2′-azinobis(3- ethylbenzthiazoline-6-sulphonate) kraft lignin depolymerization. Appl Environ Microbiol 61, 1876-1880.
    [Google Scholar]
  9. Bourbonnais, R., Paice, M. G., Leech, D. & Freiermuth, B. (1997). Reactivity and mechanism of laccase mediators for pulp deligninification. In 1997 Biological Science Symposium, pp. 335–338. Atlanta, GA: TAPPI Press.
  10. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.[CrossRef]
    [Google Scholar]
  11. Brown, M. A., Zhao, Z. & Mauk, A. G. ( 2002; ). Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor. Inorg Chim Acta 331, 232-238.[CrossRef]
    [Google Scholar]
  12. Canters, G. H. & Gilardi, G. ( 1993; ). Engineering type 1 copper sites in proteins. FEBS Lett 325, 39-48.[CrossRef]
    [Google Scholar]
  13. Cassland, P. & Jönsson, L. J. ( 1999; ). Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature. Appl Microbiol Biotechnol 52, 393-400.[CrossRef]
    [Google Scholar]
  14. Cereghino, G. P. L. & Cregg, J. M. ( 1999; ). Applications of yeasts in biotechnology: protein production and genetic analysis. Curr Opin Biotechnol 10, 422-427.[CrossRef]
    [Google Scholar]
  15. Cereghino, G. P. L. & Cregg, J. M. ( 2000; ). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24, 45-66.[CrossRef]
    [Google Scholar]
  16. Choi, G. H., Larson, T. G. & Nuss, D. L. ( 1992; ). Molecular analysis of the laccase from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol Plant–Microbe Interact 5, 119-128.[CrossRef]
    [Google Scholar]
  17. Collins, P. J., Kotterman, M. J. J., Field, J. A. & Dobson, A. D. W. ( 1996; ). Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor 290. Appl Environ Microbiol 62, 4563-4567.
    [Google Scholar]
  18. Eggert, C., Temp, U. & Eriksson, K. E. ( 1997; ). Laccase is essential for lignin degradation by the white rot fungus Pycnoporus cinnabarinus. FEBS Lett 407, 89-92.[CrossRef]
    [Google Scholar]
  19. Eggert, C., LaFayette, P. R., Temp, U., Eriksson, K. E. L. & Dean, J. F. D. ( 1998; ). Molecular analysis of a laccase gene from the white-rot fungus Pycnoporus cinnabarinus. Appl Environ Microbiol 64, 1766-1772.
    [Google Scholar]
  20. Eriksson, K. E. L., Blanchette, R. A. & Ander, P. ( 1990; ). Biochemistry of lignin degradation. In Microbial and Enzymatic Degradation of Wood and Wood Components , pp. 253-307. Edited by K. E. L. Eriksson, A. Blanchette & P. Ander. Berlin:Springer.
  21. Field, J. A., De Jong, E., Feijoo-Costa, G. & De Bont, J. A. M. ( 1993; ). Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Trends Biotechnol 58, 2219-2226.
    [Google Scholar]
  22. Gavel, Y. & Von Heijne, G. ( 1990; ). Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng 3, 433-442.[CrossRef]
    [Google Scholar]
  23. Gelo-Pujic, M., Hyug-Han, K., Butlin, N. G. & Palmore, G. T. R. ( 1999; ). Electrochemical studies of a truncated laccase produced in Pichia pastoris. Appl Environ Microbiol 65, 5515-5521.
    [Google Scholar]
  24. Ghindilis, A. L., Makeover, A., Bauer, C. G., Bier, F. F. & Scheller, F. W. ( 1995; ). Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification. Anal Chim Acta 304, 25-31.[CrossRef]
    [Google Scholar]
  25. Gianfreda, L., Xu, F. & Bollag, J.-M. ( 1999; ). Laccases: a useful group of oxidoreductive enzymes. Biorem J 3, 1-25.[CrossRef]
    [Google Scholar]
  26. Giardina, P., Cannio, R., Martirani, L., Marzullo, L., Palmieri, G. & Sannia, G. ( 1995; ). Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus. Appl Environ Microbiol 61, 2408-2413.
    [Google Scholar]
  27. Gromroff, E. D., Treiser, U. & Beck, C. F. ( 1989; ). Three light-inducible heat shock genes of Chlamydomonas reinhardtii. Mol Cell Biol 9, 3911-3918.
    [Google Scholar]
  28. Han, Y., Wilson, D. B. & Lei, X. G. ( 1999; ). Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Appl Environ Microbiol 65, 1915-1918.
    [Google Scholar]
  29. Harkin, J. M., Larsen, M. J. & Obst, J. R. ( 1974; ). Use of syringaldazine for detection of laccase in sporophores of wood rotting fungi. Mycologia 66, 469-476.[CrossRef]
    [Google Scholar]
  30. Higgins, D. G. & Sharp, P. M. ( 1989; ). Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS 5, 151-153.
    [Google Scholar]
  31. Hoshida, H., Nakao, M., Kanazawa, H., Kubo, K., Hakukawa, T., Morimasa, K., Akada, R. & Nishizawa, Y. ( 2001; ). Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterisation and expression of the yeast cDNA in yeasts. J Biosci Bioeng 92, 372-380.[CrossRef]
    [Google Scholar]
  32. Invitrogen (1998). Multi-copy Pichia Expression Kit Manual, version C. Carlsbad, CA: Invitrogen.
  33. Jönsson, L. J., Sjöström, K., Häggström, I. & Nyman, P. O. ( 1995; ). Characterization of a laccase gene from the white rot fungus Trametes versicolor and structural features of basdiomycete laccases. Biochim Biophys Acta 1251, 210-215.[CrossRef]
    [Google Scholar]
  34. Jönsson, L. J., Saloheimo, M. & Penttila, M. ( 1997; ). Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris. Curr Genet 32, 425-430.[CrossRef]
    [Google Scholar]
  35. Kawai, S., Umezawa, T. & Higuchi, T. ( 1989; ). Oxidation of methoxylated benzyl alcohols by laccase of Coriolus versicolor in the presence of syringaldahyde. Wood Res 76, 10-16.
    [Google Scholar]
  36. Kojima, Y., Tsukuda, Y., Kawai, Y., Tsukamoto, A., Sugiura, J., Sakaino, M. & Kita, Y. ( 1990; ). Cloning, sequence analysis and expression of ligninolytic polyphenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J Biol Chem 265, 15224-15230.
    [Google Scholar]
  37. Kozak, M. ( 1981; ). Possible role of flanking nucleotides in recognition of AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res 12, 857-872.
    [Google Scholar]
  38. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.[CrossRef]
    [Google Scholar]
  39. Larsson, S., Cassland, P. & Jönsson, L. J. ( 2001; ). Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in ligninocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microbiol 67, 1163-1170.[CrossRef]
    [Google Scholar]
  40. Leatham, G. F. & Stahmann, M. A. ( 1981; ). Studies on the laccase of Lentinus edodes: specificity, localization and association with the development of fruiting bodies. J Gen Microbiol 125, 147-157.
    [Google Scholar]
  41. Malkin, R. & Malmstrom, B. G. ( 1970; ). The state and function of copper in biological systems. Adv Enzymol 33, 177-244.
    [Google Scholar]
  42. Mansur, M., Suarez, T., Fernandez-Larrea, J., Brizuela, M. A. & Gonzalez, A. E. ( 1997; ). Identification of a laccase gene family in the new lignin degrading basidiomycete CECT 20197. Appl Environ Microbiol 63, 2637-2646.
    [Google Scholar]
  43. Martins, L. O., Soares, C., Pereira, M., Teixeira, M., Costa, T., Jones, G. H. & Henriques, A. O. ( 2002; ). Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277, 18849-18859.[CrossRef]
    [Google Scholar]
  44. Muñoz, C., Guillén, F., Martı́nez, A. T. & Martı́nez, M. J. ( 1997; ). Laccase isozymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation. Appl Environ Microbiol 63, 2166-2174.
    [Google Scholar]
  45. O’Callaghan, J., O’Brien, M. M., McClean, K. & Dobson, A. D. W. ( 2002; ). Optimization of the expression of a Trametes versicolor laccase gene in Pichia pastoris. J Ind Microbiol Biotechnol 29, 55-59.[CrossRef]
    [Google Scholar]
  46. Otterbein, L., Record, E., Longhi, S., Asther, M. & Moukha, S. ( 2000; ). Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur J Biochem 267, 1619-1625.[CrossRef]
    [Google Scholar]
  47. Palmieri, G., Giardina, P., Bianco, C., Scaloni, A., Capasso, A. & Sannia, G. ( 1997; ). A novel white laccase from Pleurotus ostreatus. J Biol Chem 272, 31301-31307.[CrossRef]
    [Google Scholar]
  48. Parkinson, N., Smith, I., Weaver, R. & Edwards, J. P. ( 2001; ). A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondrica. Insect Biochem Mol Biol 31, 57-63.[CrossRef]
    [Google Scholar]
  49. Potthast, A., Rosenau, T., Chen, C. L. & Gratzl, J. S. ( 1996; ). A novel method for the conversion of benzyl alcohols to benzylaldehydes by laccase-catalysed oxidation. J Mol Catal Ser A 108, 5-9.[CrossRef]
    [Google Scholar]
  50. Record, E., Punt, P. J., Chamkha, M., Labat, M., van den Hondel, C. A. M. J. J. & Asther, M. ( 2002; ). Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme. Eur J Biochem 269, 602-609.[CrossRef]
    [Google Scholar]
  51. Saloheimo, M. & Niku-Paavola, M. L. ( 1991; ). Heterologous production of a ligninolytic enzyme: Expression of the Phlebia radiata laccase gene in Trichoderma reesei. Bio/Technology 9, 987-990.[CrossRef]
    [Google Scholar]
  52. Sanchez-Amat, A., Lucas-Elı́o, P., Fernández, E., Garcı́a-Borrón, J. C. & Solano, F. ( 2001; ). Molecular cloning and functional characterisation of a unique mutipotent polyphenol oxidase from Marinomonas mediterranea. Biochim Biophys Acta 1547, 104-116.[CrossRef]
    [Google Scholar]
  53. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA Sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef]
    [Google Scholar]
  54. Schneider, P. & Pedersen, A. H. (1995). PCT. World patent application WO 95/01426.
  55. Shuttleworth, K. & Bollag, J. M. ( 1986; ). Soluble and immobilized laccase as catalysts for the transformation of substituted phenols. Enz Microb Technol 8, 171-177.[CrossRef]
    [Google Scholar]
  56. Slomczynski, D., Nakas, J. P. & Tanenbaum, S. W. ( 1995; ). Production and characterization of laccase from Botrytis cinerea 61-34. Appl Environ Microbiol 61, 907-912.
    [Google Scholar]
  57. Soden, D. M. (2002). Physiology and genetics of laccase production in Pleurotus sajor-caju. PhD thesis, National University of Ireland, Cork.
  58. Soden, D. M. & Dobson, A. D. W. ( 2001; ). Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755-1763.
    [Google Scholar]
  59. Thomas, B. R., Yonekura, M., Morgan, T. D., Czapla, T. H., Hopkins, T. L. & Kramer, K. J. ( 1989; ). A trypsin solubilised laccase from phrate pupal integument of the tobacco hornworm, Manduca sexta. Insect Biochem 19, 611-622.[CrossRef]
    [Google Scholar]
  60. Thurston, F. ( 1994; ). The structure and function of fungal laccases. Microbiology 140, 19-26.[CrossRef]
    [Google Scholar]
  61. Tomati, U., Galli, E., Di Lena, G. & Buffone, R. ( 1991; ). Induction of laccase in Pleurotus ostreatus mycelium grown in olive oil waste warers. Agrochimica 35, 275-279.
    [Google Scholar]
  62. Towbin, H., Staehelin, T. & Gordon, J. ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354.[CrossRef]
    [Google Scholar]
  63. Toyama, N. & Ogawa, K. ( 1974; ). Comparative studies on cellulolytic and oxidizing enzymes of edible and inedible wood rotters. Mushroom Sci 9, 745-760.
    [Google Scholar]
  64. Von Heijne, G. ( 1986; ). A new method for predicting signal sequence clevage sites. Nucleic Acids Res 14, 4683-4690.[CrossRef]
    [Google Scholar]
  65. Wahleithner, J. A., Xu, F., Brown, K. M., Brown, S. H., Golightly, E. J., Halkier, T., Kauppinen, S., Pederson, A. & Schneider, P. ( 1996; ). The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solan. Curr Genet 29, 395-403.[CrossRef]
    [Google Scholar]
  66. Wood, D. A. ( 1980; ). Production, purification and properties of extracellular laccase of Agaricus bisporus. J Gen Microbiol 117, 327-338.
    [Google Scholar]
  67. Xu, F., Shin, W., Brown, S. H., Wahleiter, J. A., Sundaram, U. M. & Salomon, E. I. ( 1996; ). A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability. Biochim Biophys Acta 1292, 303-311.[CrossRef]
    [Google Scholar]
  68. Yaver, D. S., Xu, F., Golightly, E. J. & 7 other authors ( 1996; ). Purification, characterization, molecular cloning and expression of two laccase genes from the white rot basidimoycete Trametes villosa. Appl Environ Microbiol 62, 834–841.
    [Google Scholar]
  69. Yaver, D. S., Del Carmen Overjero, M., Xu, F., Nelson, B. A., Brown, K. M., Halkier, T., Bernauer, S., Brown, S. H. & Kaupinen, S. ( 1999; ). Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase Lcc1. Appl Environ Microbiol 65, 4943-4948.
    [Google Scholar]
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